Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA.
Mol Cells. 2012 Nov;34(5):481-93. doi: 10.1007/s10059-012-0246-4. Epub 2012 Nov 15.
In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicating the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4(+)CD8(+) double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in γδ T cell population only in the 9.1T-NE thymus, indicating that they require a NTREGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the αβ and γδ lineage specification.
为了研究胸腺上皮细胞（TEC）亚群在 T 细胞发育中的作用，我们建立了一种新的转基因系统，能够诱导细胞特异性消融，并使用双顺反子细菌硝基还原酶和 EGFP 基因对 TEC 亚群进行标记。在转基因小鼠中，两种不同长度的 TSCOT 启动子，分别命名为 3.1T-NE 和 9.1T-NE，驱动 EGFP 在 TEC 中表达。在成年期，较小的 3.1kb TSCOT 启动子将 EGFP 表达定位在皮质，而 9.1kb 启动子则将其维持在皮质，这表明两个启动子内可能存在 TEC 特异性和隔室特异性顺式元件。在使用前体药物（如呋喃妥因和甲硝唑）诱导硝基还原酶诱导细胞死亡时，具有特异性而无旁观者杀伤作用。细胞死亡的程度取决于细胞中前体药物的剂量和胎胸腺器官培养物（FTOC）。根据 EpCAM、MHCII、CDR1 和/或 UEA-1 的表达水平分析胎胸腺基质细胞群体。EGFp 表达模式在亚群之间存在差异，表明每个 TEC 亚群的 TSCOT 启动子活性不同。FTOC 中的前体药物处理减少了总胸腺细胞和亚群的数量。在两种转基因系中，CD4(+)CD8(+)双阳性细胞群都高度敏感。令人惊讶的是，只有在 9.1T-NE 胸腺中，γδ T 细胞群明显减少，这表明它们需要一个表达 NTREGFP 的 TEC 群体。因此，这些结果支持 TEC 亚群在 αβ 和 γδ 谱系特化中的分工。
